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Exploration of Natural and Artificial Sequence Spaces: Towards a Functional Remodeling of Membrane-bound Cytochrome P450
Ist Teil von
Biocatalysis and biotransformation, 2003-01, Vol.21 (2), p.55-66
Ort / Verlag
Abingdon: Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
Erscheinungsjahr
2003
Link zum Volltext
Quelle
Taylor & Francis
Beschreibungen/Notizen
Abstract
Two complementary methods are described that associate
in vitro and in vivo steps to generate sequence
diversity by segment directed saturated mutagenesis and
family shuffling. A high-throughput DNA chip-based
procedure for the characterization and potentially the
equalization of combinatorial libraries is also presented.
Using these approaches, two combinatorial libraries of
cytochrome P450 variants derived from the CYP1A
subfamily were constructed and their sequence diversity
characterized. The results of functional screening using
high-throughput tools for the characterization of membrane
P450-catalyzed activities, suggest that the 204-214
sequence segment of human CYP1A1 is not critical for
polycyclic aromatic hydrocarbon recognition, as was
hypothesized from previous data. Moreover, mutations
in this segment do not alter the discrimination between
alkoxyresorufins, which, for all tested mutants, remained
similar to that of wild-type CYP1A1. In contrast, the
constructed CYP1A1-CYP1A2 mosaic structures, containing
multiple crossovers, exhibit a wide range of
substrate preference and regioselectivity. These mosaic
structures also discriminate between closely related
alkoxyresorufin substrates. These results open the way
to global high-throughput analysis of structure-function
relationships using combinatorial libraries of enzymes
together with libraries of structurally related substrates.